The overall reaction catalyzed by nitric oxide synthase (NOS) proceeds via two partial reactions: oxygenation of arginine to N-hydroxyarginine (NHA) (Reaction 1) requiring two electron equivalents, and oxygenation of N-hydroxyarginine to citrulline and nitric oxide (Reaction 2) requiring one electron equivalent. NADPH is the ultimate source of electrons, which are transferred to oxygen via the flavins and heme iron. Tetrahydrobiopterin (BH4) is required for NOS activity, but its possible role in electron transfer or as a reactant in the oxygenation reactions remains to be clarified. We have previously demonstrated that neuronal NOS contains a bound endogenous reductant that can substitute for NADPH in the single turnover oxygenation of arginine to NHA. During the last year we demonstrated that this reductant is a flavin semiquinone radical, and that it is potentially capable of acting as the sole source of the two electrons required for NHA synthesis: (a) oxidation of the bound flavin semiquinone follows the same first order time course as NHA synthesis, (b) two molecules of flavin semiquinone are oxidized for each molecule of NHA formed. Under the same NADPH-free reaction conditions used for measuring Reaction (1), twice as much citrulline is formed in Reaction (2) as N-hydroxyarginine formed in Reaction (1), indicating that the endogenous flavin semiquinone is also the limiting electron donor for Reaction (2). These results provide powerful support for our original thesis that BH4 does not play a redox role in nitric oxide synthase. To date, attempts at corroborating this suggestion with comparable studies of NOS-bound BH4 have been inconclusive. An automated method for BH4 determination has been developed, and should facilitate studies in this area. In order to generate the large amounts of enzyme needed for spectroscopic studies, we further optimized the E.coli expression system for neuronal NOS, and the purification of this enzyme. We now obtain highly purified NOS with a specific activity greater than those reported by others.